The 14-3-3 proteins are a family of conserved regulatory molecules expressed in all eukaryotic cells. A striking feature of the 14-3-3 proteins is their ability to bind a multitude of functionally diverse molecules. More than seventy proteins have been found to interact with 14-3-3, including kinases, phosphatases, transmembrane receptors, and transcription factors. This allows 14-3-3 to play important roles in a wide range of vital regulatory processes, such as Bad-induced apoptosis, Raf-1 -mediated cell proliferation, and Cdc25-regulated cell cycle progression. Thus, understanding the mechanisms that control the interaction of 14-3-3 with its target proteins will provide critical insight into more general questions concerning the control of intracellular signal transduction. This competing continuation application builds on our extensive experience with 14-3-3 proteins and recent new findings to test the hypothesis that one mechanism that regulates 14-3-3 function is phosphorylation of 14-3-3 proteins themselves. The main goals of the proposed research are: (i) To test the hypothesis that 14-3-3 proteins are subject to regulated phosphorylation. Phosphorylation sites of 14-3-3 isoforms will be mapped and kinases that are critical for phosphorylating these defined sites will be identified and characterized. (ii) To determine the mechanisms by which phosphorylation regulates 14-3-3/ ligand interactions. A selected inducible site will be used as a model system to test the effect of phosphorylation on 14-3-3/ ligand interactions and on 14-3-3 regulated Bad induced apoptosis. (iii) To determine and analyze the three-dimensional co-crystal structure of 14-3-3 with phosphorylated Bad protein and so provide a molecular explanation for the role of phosphorylation in 14-3-3 regulation. Because 14-3-3 often binds proteins, such as Bad, that lie at points of crosstalk between different signaling pathways, 14-3-3 binding may serve as a switch for critical cellular functions. Detailed understanding of the mechanism that controls 14-3-3/ ligand interactions may lead to novel strategies for therapeutic interventions against a variety of diseases such as human cancer.